Check out Mike Bevan's papers regarding anti-OVA T cells.
I typically DON'T use IL-2 during my initial 5 day in vitro stimulation.
I'm assuming that your EG7-OVA is expressing Class I, AND still has the
OVA gene in it. You actually might want to look at the lymph node cells
too. Do you have any experience with CTL? (i.e., CTL against simple
tumors such as FBL (H-2b), P815 (H-2d), etc...)
If you do, you probably already have the proper conditions (cell numbers
per well, media, etc...) for the in vitro phase.
You may have a problem with your immunizations. Where did you get your
immunization protocol?
Also, you may have to restimulate your "CTL" in vitro one or two more
times to obtain really efficient killers.
Without knowing what your protocols are, I cannot really pinpoint WHERE
your problems are.
In the "old" days we used to have problems generating CTL against peptides
and soluble proteins, but now days, anti-OVA CTL are fairly simple and
easy to generate.
Good Luck
Mark T. Mizuno
BMS-PRI
Seattle, Washington
Scientist at mizuno.com
In article <01bc588a$9ae71d60$060b258a at rhgf001>, "N. Sheikh"
<rhgf001 at mailrelay.qmw.ac.uk> wrote:
> Just wanted to know if anybody can help out on CTLs: am immunising mice
> (C57bl - H-2Kb) with OVA/adjuvants and assaying for CTL activity using a
> chromium release system. However cannot detect any CTL though.... I
> stimulate splenocytes with irradiated target cells (EG7-OVA) with IL-2
> (10U/ml), 5 days later retrieve cells and test for killing against EG7-OVA
> cells.
>> Does anybody have any experience with this system, any pointers.......
>> Nadeem
